EGFR-regulated EMT is a driver of vasculogenic mimicry in Nasopharyngeal Carcinoma

EGFR-regulated EMT is a driver of vasculogenic mimicry in Nasopharyngeal Carcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article EGFR-regulated EMT is a driver of vasculogenic mimicry in Nasopharyngeal Carcinoma Yue Yuan#, Yunfan Luo#, Huiru Feng#, Rui Deng, Bijun Liang, Haoran Huang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2220879/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Vascular mimicry is emerging as a potential target for anti-tumorigenesis.It involves the formation of microvascular channels composed of tumor cells. However, the mechanism of how tumor cells build into microvascular channels is not clear.The existence of a relationship between EMT and VM has been reported in the literature but the exact regulatory mechanism is unclear. Whether EMT regulates VM formation and its specific mechanism need to be further verified in NPC Materials and Methods We detected the relationship between EMT indicators and VM by immunohistochemical experiments. Also, the relationship between EMT indexes and VM indexes and clinical staging was analyzed. Cellular assays and immunoprotein blotting assays were used to detect EMT and VM changes in cells after addition of EGFR inhibitors. VM and EMT indices were examined after EGFR-targeted drug treatment in a subcutaneous tumorigenesis assay in nude mice. Conclusion EGFR-regulated EMT is a driver of vasculogenic mimicry in Nasopharyngeal Carcinoma. Nasopharyngeal carcinoma EGFR Vasculogenic mimicry EMT Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Vasculogenic mimicry (VM), the phenomenon by which tumour cells mimic ECs and form vascular channels themselves in the first reported in 1999 [ 1 – 3 ], It refers to the plasticity of invasive cancer cells to form new vascular networks, thus contributing to the perfusion of rapidly growing tumors, delivery of fluid from leaking vessels, and/or connection with constitutional endothelial layer vessels[ 4 ]. Over the next 20 years, VM has been reported in a variety of malignancies, including melanoma, glioblastoma, osteosarcoma, and hepatocellular carcinoma, as well as breast, lung, gastric, colorectal, and prostate cancers. In patients with malignancies such as breast, colorectal, prostate, liver, lung, ovarian, gastric, and bladder cancers, VM is associated with high tumor grade, invasion, metastasis, and poor prognosis[ 5 ].VM is considered an important factor in the poor anti-tumor angiogenesis effect gradually becoming a hot research topic. However, the mechanism of how tumor cells build into microvascular channels is not clear. Some studies have shown that a hypoxic and acidic microenvironment that strongly favours VM in xenografts[ 5 , 6 ]. Tumor activated platelets also possibleinduce vascular mimicry in mesenchymal stem cells and aid metastasis[ 7 ]. A study showed that microbial metabolite deoxycholic acid promotes vasculogenic mimicry formation in intestinal carcinogenesis[ 8 ]. All these factors are involved in the formation of VM. Hypoxic and acidic microenvironment is closely associated with epithelial mesenchymal transition(EMT) [ 9 – 11 ].EMT is a process in which epithelial cells, under the action of some factors, lose their cell polarity, lose their tight intercellular connections and adhesion connections, and gain the ability of infiltration and migration, becoming mesenchymal cells with morphology and characteristics[ 12 ]. Whether EMT is a VM driver raises concerns.We speculate that EMT enables tumor cells to acquire tentacles and invasive ability, which may be one of the driving factors for their formation of VM. A small amount of literature mentions the association of EMT with angiogenic mimic formation in some tumors, such as gastric cancer [ 13 ] and melanoma [ 14 ]. However, the exact relationship between EMT and VM still needs to be further clarified. Nasopharyngeal carcinoma (NPC) is a malignant tumor of nasopharynx with high affection in Southeast Asia[ 15 ]. In previous studies, we have shown that Foxq1 significantly promotes Vasculogenic mimicry (VM) formation, tumor growth, and metastasis and is effectively inhibited by EGFR inhibitors [ 16 ]. In the text we suggest that EGFR promotes the formation of VM. Whether EMT is an EGFR-regulated VM is worth further investigation. Materials And Methods Clinical samples 60 cases of nasopharyngeal carcinoma tissue samples were collected from the Southern Hospital of Southern Medical University. The research subjects selected nasopharyngeal carcinoma patients with pathologically confirmed nasopharyngeal carcinoma from 2007 to 2019. Detailed pathological, clinical data and survival time of all NPC patients were collected through outpatient and telephone follow-up. The TNM grading is based on the definition of the UICC American Joint Committee on Cancer Staging Criteria, 7th edition. Immunohistochemical And Cd31-pas Dual Staining The tissue was fixed with formalin, embedded in paraffin, and sliced at a thickness of 4 mm. After collection, the tissue was fixed with 4% paraformaldehyde at 4°C overnight. Antigen blocking was performed using 10% goat serum (AR0009, Boster, China). Anti-e-cadherin ((24E10) Rabbit mAb #3195, CST), anti-cd31 ((PECAM-1) (D8V9E) XP Rabbit mAb #77699, CST), anti-Ve-cadherin ((D87F2) XP Rabbit mAb #2500, CST) and antibodies against vimentin ((D21H3) XP Rabbit mAb #5741, CST) were incubated overnight at 4°C. DAB system (ZLI-9017, Zsbio, China) was used to detect staining. Vasculogenic mimicry structures were detected using PAS staining kit (G1281, Solarbio, China) and anti-cd31 ((PECAM-1) (D8V9E) XP Rabbit mAb #77699, CST). The number of positive cells was obtained from 5 randomly selected fields and 400x magnification. Immunohistochemical Score Immunohistochemical scoring criteria: comprehensive score = staining intensity × positive area. Staining intensity score: strong positive (3 points), positive (2 points), weak positive (1 point), negative (0 points). The proportion of positive (including strong positive) regions: 100%-76% (4 points), 75%-51% (3 points), 50%-26% (2 points), 0–25% (1 point). The above scores were averaged by two pathologists independently. Cell Culture All nasopharyngeal carcinoma cells were obtained from the Cancer Research Center of Southern Medical University, and were cultured in RPMI-1640 medium (Thermo Fisher Scientific Corporation PM15101) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific Corporation 10270- 106), 100u/ml penicillin (15140-122, Thermo Fisher Scientific, USA), 100 mg/ml streptomycin (15140-122, Thermo Fisher Scientific, USA), and humidified in 5% CO 2 The environment was maintained at 37°C. Three-dimensional Culture 24-well plates coated with 100µL Matrigel (354230, BD Biosciences, USA) reduced growth factor for each well, incubated at 37℃ for 1 h, Take 500µL medium containing 10% FBS (1*10 5 cells), spread it on the gel surface, and incubate at 37 ℃ for 24 h. Each group provides three holes. The cells were then photographed under an inverted microscope (IX71, OLYMPUS, Japan). ImageJ calculates the average number of tubular structures. Rna Isolation, Reverse Transcription, And Quantitative Realtime Pcr Total RNA was extracted from samples using RNA iso Plus (R401-01, Vazyme, China) and reverse transcribed using HiScipt III RT SuperMix for Quantitative Real-time PCR (+ gDNA wiper) (R323-01, Vazyme, China) as cDNA. Quantitative reverse transcription PCR (qRT-PCR) was performed on ABI QuantStudio5 system using ChamQ SYBR qRT-PCR Master Mix (Low ROX master mix) (Q331-02, Vazyme, China). GAPDH served as an mRNA endogenous control. All samples were normalized to an internal control and relative expression levels were calculated by using relative quantification. Western Blot Western blot The proteins extracted from samples were assayed using lysis buffer (P0013B, Beyotime, China) containing protease inhibitor cocktail (HY-K0010, MCE, USA) using radioimmunoprecipitation. Proteins were solubilized in SDS loading buffer (FD006, Fdbio, China), and the lysates were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (IPVH00010, Millipore, USA). Anti-E-Cadherin (24E10, CST, USA), Vimentin (D21H3, CST, USA), VE-Cadherin (D87F2, CST, USA), N-Cadherin (D4R1H, CST, USA), β-Catenin (D10A8, CST, USA) or GAPDH (D16H11, CST, USA) polyclonal antibodies were incubated at 4°C overnight at a dilution of 1:1000, and then incubated with species-specific enzyme-labeled secondary antibodies (1:5000 dilution) for 2 h at room temperature. Immunoreactive bands were visualized by enhanced chemiluminescence (WBKLS0100, Millipore, USA). Statistical Analyses Statistical analysis was performed using SPSS 25.0 software. All data are from at least three independent experiments. Unless otherwise stated, data are presented as SEM means. A p-value < 0.05 was considered statistically significant. Results EMT was significantly positively correlated with VM in nasopharyngeal carcinoma clinical samples. We divided the tissue samples of 60 patients with nasopharyngeal carcinoma into stage I (4 cases), stage II (8 cases), stage III (35 cases), and stage IV (13 cases). The detailed Immunohistochemical staining scores for each group are shown in Table 1 . The VM and E-cadherin and Vimentin sections stained are shown in Fig. 1 A. Table 1 The relationship between VM expression, EMT and NPC clinicopathological characteristics. Characteristic Immunohistochemical staining score n PAS + /CD31- E-cadherin vimentin Overall stage 1 I + II 3.917 ± 1.505 6.167 ± 2.167 2.500 ± 2.468 12 III + IV 8.167 ± 1.705 3.104 ± 2.146 6.625 ± 2.367 48 P value < 0.001 < 0.001 < 0.001 Tumor stage 1 T1 + T2 6.269 ± 2.662 4.654 ± 2.637 4.577 ± 2.982 26 T3 + T4 8.118 ± 1.805 3.000 ± 2.089 6.735 ± 2.478 34 P value 0.002 0.009 0.003 Node stage 1 N0 5.300 ± 3.591 5.500 ± 3.100 4.500 ± 3.837 10 N1 + N2 + N3 7.720 ± 1.863 3.360 ± 2.183 6.060 ± 2.637 50 P value 0.003 0.011 0.120 60 1.According to the 7th edition of the UICC/AJCC staging system. The association of VM and E-cadherin and Vimentin with clinical stage and TNM stage was analyzed according to immunohistochemical scores.VM indicators were higher in stage III-IV than in stage I-II nasopharyngeal carcinoma. As the clinical stage of the tumor increased, E calponin expression decreased, but vimentin increased. The results suggest that the EMT process is promoted (Fig. 1 B). Similar results were obtained in the analysis of outcomes in T-stage versus N-stage(Fig. 1 C-D). We compare the relationship between EMT and VM. E-cadherin was significantly negatively correlated with VM in nasopharyngeal carcinoma tissues (r 2 = 0.5049, p < 0.001 ), while Vimentin was positively correlated with VM (r 2 = 0.4116, p < 0.001) (Fig. 1 E-F). This indicates that EMT is significantly positively correlated with VM. The results suggest that EMT may be involved in the VM process. EMT was positively correlated with VM formation ability in vitro cell experiments. First, to investigate the effect of EGFR inhibitors on the ability of nasopharyngeal carcinoma cells to form VM, we selected two nasopharyngeal carcinoma cell lines :5-8F and CNE1. EGFR inhibitors significantly inhibit the tube-forming ability of tumor cells in 3D cell culture. The results suggest that EGFR inhibitors inhibit VM formation. (Fig. 2 A-B). Further observation of the ability of cells to form characteristic tentacles (equivalent to the occurrence of EMT markers).The results showed that after the addition of nimotuzumab, the proportion of "spindle cells" in 5-8F and CNE1 cells decreased significantly (5-8F: 66%; CNE1: 60%), indicating that the EMT process was inhibited (p < 0.05) (Fig. 2 C-D). Then, We tested the effectiveness of Nimotuzumab and found that the expression of EGFR did not change significantly in the experimental group (Nimotuzumab +) compared with the control group (Nimotuzumab -), but the content of its phosphorylated product p-EGFR was significantly decreased (Fig. 3 A). Further, Further validation of the change in EMT index after the addition of Nimotuzumab. The experimental results showed that the gene expression and protein expression of E-cadherin in two nasopharyngeal carcinoma cells increased (5-8F: 56.6%; CNE1: 49.7%), while the gene and protein expression levels of Vimentin and N-cadherin decreased (Vimentin 5-8F: 52.4%, CNE1: 37.1%; N-cadherin 5-8F: 53.3%, CNE1: 20%), while the gene expression level of β-catenin was almost unchanged (Fig. 3 B-C). We will discuss this result in detail in the Discussion section. These above results suggest the ability of EGFR inhibitors to inhibit epithelial mesenchymal transition in nasopharyngeal carcinoma. To verify whether the inhibition of EMT and VM by Nimotuzumab is related to drug concentration, we designed a Nimotuzumab drug concentration gradient experiment. The experimental results showed that with the increase of nimotuzumab concentration, the protein content and gene expression level of E-cadherin in the two cell lines gradually increased, while the protein content and gene expression level of Ve-cadherin gradually decreased (Fig. 3 D-H). In animal experiments, nimotuzumab can inhibit EMT process and VM formation. In our previous study, in order to investigate the relationship between vasculogenic mimicry and Foxq1 and EGFR, we performed subcutaneous tumorigenesis experiments[ 1 ]. Brief description of the experiment: 5-8F cells were injected subcutaneously in nude mice. The tail vein of the experimental group was injected with Nimotuzumab and the tail vein of the control group was injected with saline after tumor formation. We re-sliced and stained the tumor specimens utilizing previous animal experiments from our experimental group. First, PAS/CD31 double staining was performed to detect the formation of vasculogenic mimicry in tumor tissues. We found that the tumor tissues of mice injected with nimotuzumab in the tail vein were less prone to vasculogenic mimicry than those of mice injected with normal saline. (Fig. 4 A-B). Next, we examined EMT-related indicators (E-cadherin, Vimentin) and VM related indicators (VE-cadherin) in these animal tissues. We found that e-cadherin expression was decreased and Vimentin and VE-cadherin expression was increased in nimotuzumab treated tumor tissues compared with saline treated mouse tumor tissues. Nimotuzumab inhibited the EMT process and VM suppression was also observed. (Fig. 4 C-F). Discussion In this article, we verified the relationship between angiogenic mimicry and EMT from tissue samples, cellular experiments and animal experiments. The results suggest that EMT may act as a bridge mediating the EGFR pathway and promote angiogenic mimicry in nasopharyngeal carcinoma. Vasculogenic mimicry (VM) is a vascular-like structure which can mimic the embryonic vascular network pattern to nourish the tumour tissue[ 17 ]. As a unique perfusion way, VM is correlated with tumour progression, invasion, metastasis and lower 5-year survival rate. Notably, epithelial-mesenchymal transition (EMT) regulators and EMT-related transcription factors are highly up-regulated in VM-forming tumour cells, which demonstrated that EMT may play a crucial role in VM formation[ 18 ]. Therefore, the up-regulation of EMT-associated adhesion molecules and other factors can also make a contribution in VM-forming process[ 19 ]. Our study found a correlation between EMT and vasculogenic mimicry in nasopharyngeal carcinoma. When we inhibited the formation of vasculogenic mimicry vessels in nasopharyngeal carcinoma by using EGFR inhibitors, we detected that the EMT process was inhibited. Our results found a strong correlation between EMT and VM. it is highly likely that EMT is a bridge for VM formation. However, experiments are still needed to confirm their direct relationship. We also added that the EGFR signaling pathway can promote the formation of EMT and VM. During the experiment, we discovered an interesting phenomenon. we began to extract the total protein of the cells to detect the expression of β-catenin. There was no difference in the expression of β-catenin between the nimotuzumab-treated group and the negative control group. Then we extracted the β-catenin expression of the cell nucleus, and the beta-catenin expression of the nimotuzumab treatment group decreased (Additional Fig. 1 ). β-catenin is a multifunctional protein that helps cells respond to signals and influences outside the cell by interacting with the cytoskeleton. This protein acts as a transcription factor in the nucleus and turns on genes that promote cell division. In the absence of Wnt signaling, β-catenin is degraded by protein complexes including Axin, APC, Ser/Thr kinases GSK-3 and CK1, protein phosphatase 2A (PP2A), and E3-ubiquitin ligase B-TrCP. This complex specifies the B-TrCP recognition site on β-catenin by phosphorylation of a conserved Ser/Thrrich sequence near the amino terminal. Phosphorylation requires Axin to scaffold GSK-3 and CK1 and β-catenin. After phosphorylation and ubiquitination, β-catenin is degraded by the proteasome. Binding of Wnt to its receptor induces binding of Axin to phosphorylated lipoprotein receptor-associated protein (LRP). The breakdown of the complex stabilizes β-catenin, which accumulates in the cytoplasm and enters the nucleus, where it subsequently binds to TCF in the nucleus, thereby upregulation of target genes[ 20 ]. In this study, western blot analysis showed that the level of β-catenin in the nucleus decreased (with histone H3 as internal reference) when vasculogenic mimicry formation was inhibited. However, qRT-PCR showed no change in the expression level of the CTNNB1 gene, which regulates the expression of β-catenin. Therefore, we hypothesized that during EMT, the total amount of β-catenin did not change significantly, but its intracellular distribution changed, from cytoplasm to nucleus. The mechanism of β-catenin regulation of EMT and VM remains to be further studied. Conclusions EGFR-regulated EMT is a driver of vasculogenic mimicry in Nasopharyngeal Carcinoma. Declarations Contributions : (I) Conception and design: X Peng, X Liu; (II) Administrative support: X Liu, B Yu; (III) Provision of study materials or patients: Y Lou; (IV) Collection and assembly of data: Y Yue, H Feng; (V) Data analysis and interpretation: Y Yue; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors. Ethics approval All clinical studies were approved by the Ethics Committee of Southern Medical University (Ethnical approval No. NFEC-2017-165). The study protocol is performed in accordance with the relevant guidelines. This study informed consent was obtained from all subjects and/or their legal guardian(s). Consent for publication Not applicable. Conflicts of Interest The authors declare no competing interests. Data availability The data that support the findings of this study are available from the corresponding author upon reasonable request. Funding This study was supported by grants from the National Natural Science Foundation of China (81702696), Natural Science Foundation of Guangdong Province of China (2017A030310040 and 2020A1515010176), and Supported by Beijing xisike Clinical Oncology Research Foundation. Authors' contributions Y.Y. and P.X.H. designed the study. Y.Y., L.Y.F., F.H.R. and Y.B.L. performed the work. Y.Y., L.X. and P.X.H. performed the statistical analysis. D.R., L.B.J., H.H.R. and Y.X.F reviewed the data. All authors discussed the data. Y.Y, P.X.H, Y.B.L and L.X.drafted the manuscript. All co-authors read and approved the manuscript. Acknowledgements Thanks to BIOTECH PHARMACEUTICAL CO.LTD for providing Nimotuzumab injection for the experiment. References Hernández De La Cruz, O.N., et al., Regulation Networks Driving Vasculogenic Mimicry in Solid Tumors. Frontiers in Oncology, 2020. 9. Krishna Priya, S., et al., Tumour angiogenesis-Origin of blood vessels. International Journal of Cancer, 2016. 139(4): p. 729-735. Maniotis, A.J., et al., Vascular channel formation by human melanoma cells in vivo and in vitro: vasculogenic mimicry. Am J Pathol, 1999. 155(3): p. 739-52. Seftor, R.E.B., et al., Tumor Cell Vasculogenic Mimicry. The American Journal of Pathology, 2012. 181(4): p. 1115-1125. Andreucci, E., et al., Physicochemical aspects of the tumour microenvironment as drivers of vasculogenic mimicry. Cancer and Metastasis Reviews, 2022. Lezcano, C., et al., Merkel cell carcinoma expresses vasculogenic mimicry: demonstration in patients and experimental manipulation in xenografts. Lab Invest, 2014. 94(10): p. 1092-102. Bhuniya, A., et al., Tumor activated platelets induce vascular mimicry in mesenchymal stem cells and aid metastasis. Cytokine, 2022. 158: p. 155998. Song, X., et al., Microbial metabolite deoxycholic acid promotes vasculogenic mimicry formation in intestinal carcinogenesis. Cancer Science, 2022. 113(2): p. 459-477. Carroll, C.P., et al., Targeting hypoxia regulated sodium driven bicarbonate transporters reduces triple negative breast cancer metastasis. Neoplasia, 2022. 25: p. 41-52. Riemann, A., et al., Extracellular Acidosis Modulates the Expression of Epithelial-Mesenchymal Transition (EMT) Markers and Adhesion of Epithelial and Tumor Cells. Neoplasia, 2019. 21(5): p. 450-458. Riemann, A., et al., The Acidic Tumor Microenvironment Affects Epithelial-Mesenchymal Transition Markers as Well as Adhesion of NCI-H358 Lung Cancer Cells. Adv Exp Med Biol, 2021. 1269: p. 179-183. Lamouille, S., J. Xu and R. Derynck, Molecular mechanisms of epithelial–mesenchymal transition. Nature Reviews Molecular Cell Biology, 2014. 15(3): p. 178-196. Zhao, J., et al., LncRNA PVT1 induces aggressive vasculogenic mimicry formation through activating the STAT3/Slug axis and epithelial-to-mesenchymal transition in gastric cancer. Cellular Oncology, 2020. 43(5): p. 863-876. Li, W. and Y. Zhou, LRIG1 acts as a critical regulator of melanoma cell invasion, migration, and vasculogenic mimicry upon hypoxia by regulating EGFR/ERK-triggered epithelial–mesenchymal transition. Bioscience Reports, 2019. 39(1). Chen, Y.P., et al., Nasopharyngeal carcinoma. Lancet, 2019. 394(10192): p. 64-80. Luo, Y., et al., Foxq1 promotes metastasis of nasopharyngeal carcinoma by inducing vasculogenic mimicry via the EGFR signaling pathway. Cell Death & Disease, 2021. 12(5). Luo, Q., et al., Vasculogenic mimicry in carcinogenesis and clinical applications. Journal of Hematology & Oncology, 2020. 13(1). Huang, Y., W. Hong and X. Wei, The molecular mechanisms and therapeutic strategies of EMT in tumor progression and metastasis. Journal of Hematology & Oncology, 2022. 15(1). Liu, Q., et al., The relationship between vasculogenic mimicry and epithelial-mesenchymal transitions. Journal of Cellular and Molecular Medicine, 2016. 20(9): p. 1761-1769. Nusse, R. and H. Clevers, Wnt/beta-Catenin Signaling, Disease, and Emerging Therapeutic Modalities. Cell, 2017. 169(6): p. 985-999. Luo Y, Wang J, Wang F, Liu X, Lu J, Yu X, Ma X, Peng X, Li X: Foxq1 promotes metastasis of nasopharyngeal carcinoma by inducing vasculogenic mimicry via the EGFR signaling pathway . Cell death & disease 2021, 12 (5):411 Additional Declarations No competing interests reported. Supplementary Files AdditionalFigureandlegend.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2220879","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":152873981,"identity":"d44978ff-a33e-4d27-ab21-a9da70c9f393","order_by":0,"name":"Yue Yuan#","email":"","orcid":"","institution":"Nanfang Hospital, Southern Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Yuan#","suffix":""},{"id":152873984,"identity":"1ffb222f-3138-4964-9da3-9272fcbc85d2","order_by":1,"name":"Yunfan Luo#","email":"","orcid":"","institution":"Shenzhen Second People's Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yunfan","middleName":"","lastName":"Luo#","suffix":""},{"id":152873987,"identity":"77b6de9c-6a2f-4b0a-98d2-286008ff9900","order_by":2,"name":"Huiru Feng#","email":"","orcid":"","institution":"Nanfang Hospital, Southern Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Huiru","middleName":"","lastName":"Feng#","suffix":""},{"id":152873989,"identity":"cea2a50b-c020-4312-a83f-b75653cbcb1b","order_by":3,"name":"Rui Deng","email":"","orcid":"","institution":"Nanfang Hospital, Southern Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rui","middleName":"","lastName":"Deng","suffix":""},{"id":152873990,"identity":"ddef5ba1-b752-45de-83ad-ffda5dfb6e7c","order_by":4,"name":"Bijun Liang","email":"","orcid":"","institution":"Nanfang Hospital, Southern Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Bijun","middleName":"","lastName":"Liang","suffix":""},{"id":152873991,"identity":"a68aabfa-00ab-40d5-8e56-cb03966a48ec","order_by":5,"name":"Haoran Huang","email":"","orcid":"","institution":"Nanfang Hospital, Southern Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Haoran","middleName":"","lastName":"Huang","suffix":""},{"id":152873995,"identity":"79463c70-4465-4c56-b961-1ad303ae531a","order_by":6,"name":"Xiaofei Yuan","email":"","orcid":"","institution":"Nanfang Hospital, Southern Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaofei","middleName":"","lastName":"Yuan","suffix":""},{"id":152873996,"identity":"fa523cde-8e0b-40da-b65d-cc81d733004e","order_by":7,"name":"Zilu Chen","email":"","orcid":"","institution":"Nanfang Hospital, Southern Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zilu","middleName":"","lastName":"Chen","suffix":""},{"id":152873998,"identity":"54e11e7b-c07f-4383-aee1-2d666272b557","order_by":8,"name":"Bolong Yu","email":"","orcid":"","institution":"Nanfang Hospital, Southern Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Bolong","middleName":"","lastName":"Yu","suffix":""},{"id":152874001,"identity":"4ed1524b-527e-4c24-8730-3d1d1e88ec6e","order_by":9,"name":"Xiong Liu","email":"","orcid":"","institution":"Nanfang Hospital, Southern Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiong","middleName":"","lastName":"Liu","suffix":""},{"id":152874002,"identity":"77a66383-32ce-4ba2-8f9e-30d52c512844","order_by":10,"name":"Xiaohong Peng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIie3RMQrCMBTG8YRAujz3V3qJFKE4FDyIS6DQSSfXgoEOjl0teImewMoDXXqAjoqrg15ADLi4Nd0E85/zI3wJYz7fD8ZLcbw8X7g5nI0jEVuZxTuZctO1jiSoIIlA5tz02pFMSplNAUjw+tb0rEgXgyS0W657JCmifD1jp3xlhkhsb1F3RSCjZYLc0DCZEyQImhDCzpHwEpIQ2lwhgjOxj1ybVCuwW7TLFl6R/UqDWgXU9I8iHSbfKdRjjn/IWOHz+Xz/0Rtnqz3e4cDguwAAAABJRU5ErkJggg==","orcid":"","institution":"Nanfang Hospital, Southern Medical University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Xiaohong","middleName":"","lastName":"Peng","suffix":""}],"badges":[],"createdAt":"2022-10-31 08:29:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2220879/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2220879/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":29338166,"identity":"1feb9d08-d658-421e-829f-eb493c851b62","added_by":"auto","created_at":"2022-11-21 15:31:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1365412,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of EMT-related factors and vasculogenic mimicry in clinical nasopharyngeal carcinoma A \u003c/strong\u003eComparison of the expression levels of VM, E-cadherin and Vimentin in nasopharyngeal carcinoma tissues of different stages (I+II, III+IV). Left: 100x field of view; right: 400x field of view; Scale bars represent 50 μm. \u003cstrong\u003eB-D\u003c/strong\u003eThe relationship between immunohistochemical indexes PAS+/CD31, E-cadherin, Vimentin and clinical stage, T stage and N stage. \u003cstrong\u003eE-F \u003c/strong\u003eSpearman correlation analysis of E-cadherin, Vimentin and VM in nasopharyngeal carcinoma tissue.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-2220879/v1/2ac957bb30e642ad8376236d.png"},{"id":29338165,"identity":"dd01624d-3a89-4680-8c85-146dea8c0cd4","added_by":"auto","created_at":"2022-11-21 15:31:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1305869,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEGFR inhibitors inhibit VM formation in nasopharyngeal carcinoma cells. A-B\u003c/strong\u003e 3D culture, the tube-forming ability of the experimental group (Nimotuzumab +) was significantly decreased compared with the control group (Nimotuzumab -); ×200 field of view. \u003cstrong\u003eC-D\u003c/strong\u003e Cell morphology was observed under an inverted microscope. The number of \"spindle cells\" (framed by dashed line) was significantly reduced in the experimental group (Nimotuzumab +) compared to the control group (Nimotuzumab -); 200x field of view.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-2220879/v1/3d02e0801daabc342b38d536.png"},{"id":29339433,"identity":"ff11d892-932a-4a48-9bae-760a17015d59","added_by":"auto","created_at":"2022-11-21 15:39:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":388388,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe relationship between EMT and VM in nasopharyngeal carcinoma cells in vitro. A. \u003c/strong\u003eWestern blot experiments were performed to detect the expression of EGFR and its phosphorylation product p-EGFR in 5-8F and CNE1 cell lines. The expression level of EGFR was almost unchanged, and the expression level of p-EGFR decreased significantly.\u003cstrong\u003e B-C \u003c/strong\u003eqRT-PCR experiment was used to detect the gene expression levels of E-cadherin, N-cadherin, Vimentin and β-catenin in two nasopharyngeal carcinoma cell lines before and after adding Nimotuzumab.\u003cstrong\u003e D-E \u003c/strong\u003eAs a result of Western blot experiment, the contents of E-cadherin, N-cadherin, Vimentin and nuclear protein β-catenin in total protein were detected. \u003cstrong\u003eF-G\u003c/strong\u003e In the drug concentration gradient experiment, with the increase of nimotuzumab concentration, the protein content and gene expression level of E-cadherin in the two cell lines gradually increased, while the protein content and gene expression level of Ve-cadherin gradually decreased; p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-2220879/v1/e9b38726b1a526bc144f385f.png"},{"id":29338169,"identity":"5ca216ec-9286-4ae6-99b3-855739aecf8e","added_by":"auto","created_at":"2022-11-21 15:31:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1060420,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe relationship between EMT and VM in nasopharyngeal carcinoma tissue in vivo animal experiment.\u003c/strong\u003e \u003cstrong\u003eA-B\u003c/strong\u003ePAS/CD31 double staining was used to compare the formation of VM in the two groups. (Magnification, 200) scale means 50 μm. \u003cstrong\u003eC-F\u003c/strong\u003e Immunohistochemical staining was used to evaluate the expression levels of EMT and VM related indexes in the two groups; p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-2220879/v1/302fe7fc85268ec765a6559b.png"},{"id":30201406,"identity":"d4932689-9a50-4eb1-929c-7a927ea3b813","added_by":"auto","created_at":"2022-12-12 13:14:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2746456,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2220879/v1/cc7c9e56-4f6f-4650-bf65-69a547921ce3.pdf"},{"id":29338168,"identity":"f29452a8-d638-4c26-abe8-c622f29cd1e8","added_by":"auto","created_at":"2022-11-21 15:31:41","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":154808,"visible":true,"origin":"","legend":"","description":"","filename":"AdditionalFigureandlegend.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2220879/v1/dd20b0c0b144539985056ecc.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"EGFR-regulated EMT is a driver of vasculogenic mimicry in Nasopharyngeal Carcinoma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eVasculogenic mimicry (VM), the phenomenon by which tumour cells mimic ECs and form vascular channels themselves in the first reported in 1999 [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], It refers to the plasticity of invasive cancer cells to form new vascular networks, thus contributing to the perfusion of rapidly growing tumors, delivery of fluid from leaking vessels, and/or connection with constitutional endothelial layer vessels[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Over the next 20 years, VM has been reported in a variety of malignancies, including melanoma, glioblastoma, osteosarcoma, and hepatocellular carcinoma, as well as breast, lung, gastric, colorectal, and prostate cancers. In patients with malignancies such as breast, colorectal, prostate, liver, lung, ovarian, gastric, and bladder cancers, VM is associated with high tumor grade, invasion, metastasis, and poor prognosis[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].VM is considered an important factor in the poor anti-tumor angiogenesis effect gradually becoming a hot research topic. However, the mechanism of how tumor cells build into microvascular channels is not clear.\u003c/p\u003e \u003cp\u003eSome studies have shown that a hypoxic and acidic microenvironment that strongly favours VM in xenografts[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Tumor activated platelets also possibleinduce vascular mimicry in mesenchymal stem cells and aid metastasis[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. A study showed that microbial metabolite deoxycholic acid promotes vasculogenic mimicry formation in intestinal carcinogenesis[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. All these factors are involved in the formation of VM. Hypoxic and acidic microenvironment is closely associated with epithelial mesenchymal transition(EMT) [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].EMT is a process in which epithelial cells, under the action of some factors, lose their cell polarity, lose their tight intercellular connections and adhesion connections, and gain the ability of infiltration and migration, becoming mesenchymal cells with morphology and characteristics[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Whether EMT is a VM driver raises concerns.We speculate that EMT enables tumor cells to acquire tentacles and invasive ability, which may be one of the driving factors for their formation of VM. A small amount of literature mentions the association of EMT with angiogenic mimic formation in some tumors, such as gastric cancer [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and melanoma [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, the exact relationship between EMT and VM still needs to be further clarified.\u003c/p\u003e \u003cp\u003eNasopharyngeal carcinoma (NPC) is a malignant tumor of nasopharynx with high affection in Southeast Asia[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In previous studies, we have shown that Foxq1 significantly promotes Vasculogenic mimicry (VM) formation, tumor growth, and metastasis and is effectively inhibited by EGFR inhibitors [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In the text we suggest that EGFR promotes the formation of VM. Whether EMT is an EGFR-regulated VM is worth further investigation.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eClinical samples\u003c/h2\u003e \u003cp\u003e60 cases of nasopharyngeal carcinoma tissue samples were collected from the Southern Hospital of Southern Medical University. The research subjects selected nasopharyngeal carcinoma patients with pathologically confirmed nasopharyngeal carcinoma from 2007 to 2019. Detailed pathological, clinical data and survival time of all NPC patients were collected through outpatient and telephone follow-up. The TNM grading is based on the definition of the UICC American Joint Committee on Cancer Staging Criteria, 7th edition.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunohistochemical And Cd31-pas Dual Staining\u003c/h3\u003e\n\u003cp\u003eThe tissue was fixed with formalin, embedded in paraffin, and sliced at a thickness of 4 mm. After collection, the tissue was fixed with 4% paraformaldehyde at 4\u0026deg;C overnight. Antigen blocking was performed using 10% goat serum (AR0009, Boster, China). Anti-e-cadherin ((24E10) Rabbit mAb #3195, CST), anti-cd31 ((PECAM-1) (D8V9E) XP Rabbit mAb #77699, CST), anti-Ve-cadherin ((D87F2) XP Rabbit mAb #2500, CST) and antibodies against vimentin ((D21H3) XP Rabbit mAb #5741, CST) were incubated overnight at 4\u0026deg;C. DAB system (ZLI-9017, Zsbio, China) was used to detect staining. Vasculogenic mimicry structures were detected using PAS staining kit (G1281, Solarbio, China) and anti-cd31 ((PECAM-1) (D8V9E) XP Rabbit mAb #77699, CST). The number of positive cells was obtained from 5 randomly selected fields and 400x magnification.\u003c/p\u003e\n\u003ch3\u003eImmunohistochemical Score\u003c/h3\u003e\n\u003cp\u003eImmunohistochemical scoring criteria: comprehensive score\u0026thinsp;=\u0026thinsp;staining intensity \u0026times; positive area. Staining intensity score: strong positive (3 points), positive (2 points), weak positive (1 point), negative (0 points). The proportion of positive (including strong positive) regions: 100%-76% (4 points), 75%-51% (3 points), 50%-26% (2 points), 0\u0026ndash;25% (1 point). The above scores were averaged by two pathologists independently.\u003c/p\u003e\n\u003ch3\u003eCell Culture\u003c/h3\u003e\n\u003cp\u003eAll nasopharyngeal carcinoma cells were obtained from the Cancer Research Center of Southern Medical University, and were cultured in RPMI-1640 medium (Thermo Fisher Scientific Corporation PM15101) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific Corporation 10270- 106), 100u/ml penicillin (15140-122, Thermo Fisher Scientific, USA), 100 mg/ml streptomycin (15140-122, Thermo Fisher Scientific, USA), and humidified in 5% CO\u003csub\u003e2\u003c/sub\u003e The environment was maintained at 37\u0026deg;C.\u003c/p\u003e\n\u003ch3\u003eThree-dimensional Culture\u003c/h3\u003e\n\u003cp\u003e24-well plates coated with 100\u0026micro;L Matrigel (354230, BD Biosciences, USA) reduced growth factor for each well, incubated at 37℃ for 1 h, Take 500\u0026micro;L medium containing 10% FBS (1*10\u003csup\u003e5\u003c/sup\u003e cells), spread it on the gel surface, and incubate at 37 ℃ for 24 h. Each group provides three holes. The cells were then photographed under an inverted microscope (IX71, OLYMPUS, Japan). ImageJ calculates the average number of tubular structures.\u003c/p\u003e\n\u003ch3\u003eRna Isolation, Reverse Transcription, And Quantitative Realtime Pcr\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from samples using RNA iso Plus (R401-01, Vazyme, China) and reverse transcribed using HiScipt III RT SuperMix for Quantitative Real-time PCR (+\u0026thinsp;gDNA wiper) (R323-01, Vazyme, China) as cDNA. Quantitative reverse transcription PCR (qRT-PCR) was performed on ABI QuantStudio5 system using ChamQ SYBR qRT-PCR Master Mix (Low ROX master mix) (Q331-02, Vazyme, China). GAPDH served as an mRNA endogenous control. All samples were normalized to an internal control and relative expression levels were calculated by using relative quantification.\u003c/p\u003e\n\u003ch3\u003eWestern Blot\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eWestern blot\u003c/div\u003e \u003cp\u003eThe proteins extracted from samples were assayed using lysis buffer (P0013B, Beyotime, China) containing protease inhibitor cocktail (HY-K0010, MCE, USA) using radioimmunoprecipitation. Proteins were solubilized in SDS loading buffer (FD006, Fdbio, China), and the lysates were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (IPVH00010, Millipore, USA). Anti-E-Cadherin (24E10, CST, USA), Vimentin (D21H3, CST, USA), VE-Cadherin (D87F2, CST, USA), N-Cadherin (D4R1H, CST, USA), β-Catenin (D10A8, CST, USA) or GAPDH (D16H11, CST, USA) polyclonal antibodies were incubated at 4\u0026deg;C overnight at a dilution of 1:1000, and then incubated with species-specific enzyme-labeled secondary antibodies (1:5000 dilution) for 2 h at room temperature. Immunoreactive bands were visualized by enhanced chemiluminescence (WBKLS0100, Millipore, USA).\u003c/p\u003e\n\u003ch3\u003eStatistical Analyses\u003c/h3\u003e\n\u003cp\u003eStatistical analysis was performed using SPSS 25.0 software. All data are from at least three independent experiments. Unless otherwise stated, data are presented as SEM means. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eEMT was significantly positively correlated with VM in nasopharyngeal carcinoma clinical samples.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe divided the tissue samples of 60 patients with nasopharyngeal carcinoma into stage I (4 cases), stage II (8 cases), stage III (35 cases), and stage IV (13 cases). The detailed Immunohistochemical staining scores for each group are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The VM and E-cadherin and Vimentin sections stained are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe relationship between VM expression, EMT and NPC clinicopathological characteristics.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCharacteristic\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eImmunohistochemical staining score\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePAS\u003csup\u003e+\u003c/sup\u003e/CD31-\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eE-cadherin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003evimentin\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOverall stage\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI\u0026thinsp;+\u0026thinsp;II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.917\u0026thinsp;\u0026plusmn;\u0026thinsp;1.505\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.167\u0026thinsp;\u0026plusmn;\u0026thinsp;2.167\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.500\u0026thinsp;\u0026plusmn;\u0026thinsp;2.468\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIII\u0026thinsp;+\u0026thinsp;IV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.167\u0026thinsp;\u0026plusmn;\u0026thinsp;1.705\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.104\u0026thinsp;\u0026plusmn;\u0026thinsp;2.146\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.625\u0026thinsp;\u0026plusmn;\u0026thinsp;2.367\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor stage\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT1\u0026thinsp;+\u0026thinsp;T2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.269\u0026thinsp;\u0026plusmn;\u0026thinsp;2.662\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.654\u0026thinsp;\u0026plusmn;\u0026thinsp;2.637\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.577\u0026thinsp;\u0026plusmn;\u0026thinsp;2.982\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT3\u0026thinsp;+\u0026thinsp;T4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.118\u0026thinsp;\u0026plusmn;\u0026thinsp;1.805\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.000\u0026thinsp;\u0026plusmn;\u0026thinsp;2.089\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.735\u0026thinsp;\u0026plusmn;\u0026thinsp;2.478\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNode stage\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.300\u0026thinsp;\u0026plusmn;\u0026thinsp;3.591\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.500\u0026thinsp;\u0026plusmn;\u0026thinsp;3.100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.500\u0026thinsp;\u0026plusmn;\u0026thinsp;3.837\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN1\u0026thinsp;+\u0026thinsp;N2\u0026thinsp;+\u0026thinsp;N3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.720\u0026thinsp;\u0026plusmn;\u0026thinsp;1.863\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.360\u0026thinsp;\u0026plusmn;\u0026thinsp;2.183\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.060\u0026thinsp;\u0026plusmn;\u0026thinsp;2.637\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e1.According to the 7th edition of the UICC/AJCC staging system.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe association of VM and E-cadherin and Vimentin with clinical stage and TNM stage was analyzed according to immunohistochemical scores.VM indicators were higher in stage III-IV than in stage I-II nasopharyngeal carcinoma. As the clinical stage of the tumor increased, E calponin expression decreased, but vimentin increased. The results suggest that the EMT process is promoted (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Similar results were obtained in the analysis of outcomes in T-stage versus N-stage(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-D).\u003c/p\u003e \u003cp\u003eWe compare the relationship between EMT and VM. E-cadherin was significantly negatively correlated with VM in nasopharyngeal carcinoma tissues (r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.5049, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e), while Vimentin was positively correlated with VM (r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.4116, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-F). This indicates that EMT is significantly positively correlated with VM. The results suggest that EMT may be involved in the VM process.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEMT was positively correlated with VM formation ability in vitro cell experiments.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFirst, to investigate the effect of EGFR inhibitors on the ability of nasopharyngeal carcinoma cells to form VM, we selected two nasopharyngeal carcinoma cell lines :5-8F and CNE1. EGFR inhibitors significantly inhibit the tube-forming ability of tumor cells in 3D cell culture. The results suggest that EGFR inhibitors inhibit VM formation. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). Further observation of the ability of cells to form characteristic tentacles (equivalent to the occurrence of EMT markers).The results showed that after the addition of nimotuzumab, the proportion of \"spindle cells\" in 5-8F and CNE1 cells decreased significantly (5-8F: 66%; CNE1: 60%), indicating that the EMT process was inhibited (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThen, We tested the effectiveness of Nimotuzumab and found that the expression of EGFR did not change significantly in the experimental group (Nimotuzumab +) compared with the control group (Nimotuzumab -), but the content of its phosphorylated product p-EGFR was significantly decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurther, Further validation of the change in EMT index after the addition of Nimotuzumab. The experimental results showed that the gene expression and protein expression of E-cadherin in two nasopharyngeal carcinoma cells increased (5-8F: 56.6%; CNE1: 49.7%), while the gene and protein expression levels of Vimentin and N-cadherin decreased (Vimentin 5-8F: 52.4%, CNE1: 37.1%; N-cadherin 5-8F: 53.3%, CNE1: 20%), while the gene expression level of β-catenin was almost unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-C). We will discuss this result in detail in the \u003cspan refid=\"Sec12\" class=\"InternalRef\"\u003eDiscussion\u003c/span\u003e section. These above results suggest the ability of EGFR inhibitors to inhibit epithelial mesenchymal transition in nasopharyngeal carcinoma.\u003c/p\u003e \u003cp\u003eTo verify whether the inhibition of EMT and VM by Nimotuzumab is related to drug concentration, we designed a Nimotuzumab drug concentration gradient experiment. The experimental results showed that with the increase of nimotuzumab concentration, the protein content and gene expression level of E-cadherin in the two cell lines gradually increased, while the protein content and gene expression level of Ve-cadherin gradually decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-H).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn animal experiments, nimotuzumab can inhibit EMT process and VM formation.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn our previous study, in order to investigate the relationship between vasculogenic mimicry and Foxq1 and EGFR, we performed subcutaneous tumorigenesis experiments[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Brief description of the experiment: 5-8F cells were injected subcutaneously in nude mice. The tail vein of the experimental group was injected with Nimotuzumab and the tail vein of the control group was injected with saline after tumor formation. We re-sliced and stained the tumor specimens utilizing previous animal experiments from our experimental group.\u003c/p\u003e \u003cp\u003eFirst, PAS/CD31 double staining was performed to detect the formation of vasculogenic mimicry in tumor tissues. We found that the tumor tissues of mice injected with nimotuzumab in the tail vein were less prone to vasculogenic mimicry than those of mice injected with normal saline. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B). Next, we examined EMT-related indicators (E-cadherin, Vimentin) and VM related indicators (VE-cadherin) in these animal tissues. We found that e-cadherin expression was decreased and Vimentin and VE-cadherin expression was increased in nimotuzumab treated tumor tissues compared with saline treated mouse tumor tissues. Nimotuzumab inhibited the EMT process and VM suppression was also observed. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this article, we verified the relationship between angiogenic mimicry and EMT from tissue samples, cellular experiments and animal experiments. The results suggest that EMT may act as a bridge mediating the EGFR pathway and promote angiogenic mimicry in nasopharyngeal carcinoma.\u003c/p\u003e \u003cp\u003eVasculogenic mimicry (VM) is a vascular-like structure which can mimic the embryonic vascular network pattern to nourish the tumour tissue[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. As a unique perfusion way, VM is correlated with tumour progression, invasion, metastasis and lower 5-year survival rate. Notably, epithelial-mesenchymal transition (EMT) regulators and EMT-related transcription factors are highly up-regulated in VM-forming tumour cells, which demonstrated that EMT may play a crucial role in VM formation[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Therefore, the up-regulation of EMT-associated adhesion molecules and other factors can also make a contribution in VM-forming process[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Our study found a correlation between EMT and vasculogenic mimicry in nasopharyngeal carcinoma. When we inhibited the formation of vasculogenic mimicry vessels in nasopharyngeal carcinoma by using EGFR inhibitors, we detected that the EMT process was inhibited. Our results found a strong correlation between EMT and VM. it is highly likely that EMT is a bridge for VM formation. However, experiments are still needed to confirm their direct relationship. We also added that the EGFR signaling pathway can promote the formation of EMT and VM.\u003c/p\u003e \u003cp\u003eDuring the experiment, we discovered an interesting phenomenon. we began to extract the total protein of the cells to detect the expression of β-catenin. There was no difference in the expression of β-catenin between the nimotuzumab-treated group and the negative control group. Then we extracted the β-catenin expression of the cell nucleus, and the beta-catenin expression of the nimotuzumab treatment group decreased (Additional Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). β-catenin is a multifunctional protein that helps cells respond to signals and influences outside the cell by interacting with the cytoskeleton. This protein acts as a transcription factor in the nucleus and turns on genes that promote cell division. In the absence of Wnt signaling, β-catenin is degraded by protein complexes including Axin, APC, Ser/Thr kinases GSK-3 and CK1, protein phosphatase 2A (PP2A), and E3-ubiquitin ligase B-TrCP. This complex specifies the B-TrCP recognition site on β-catenin by phosphorylation of a conserved Ser/Thrrich sequence near the amino terminal. Phosphorylation requires Axin to scaffold GSK-3 and CK1 and β-catenin. After phosphorylation and ubiquitination, β-catenin is degraded by the proteasome. Binding of Wnt to its receptor induces binding of Axin to phosphorylated lipoprotein receptor-associated protein (LRP). The breakdown of the complex stabilizes β-catenin, which accumulates in the cytoplasm and enters the nucleus, where it subsequently binds to TCF in the nucleus, thereby upregulation of target genes[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In this study, western blot analysis showed that the level of β-catenin in the nucleus decreased (with histone H3 as internal reference) when vasculogenic mimicry formation was inhibited. However, qRT-PCR showed no change in the expression level of the CTNNB1 gene, which regulates the expression of β-catenin. Therefore, we hypothesized that during EMT, the total amount of β-catenin did not change significantly, but its intracellular distribution changed, from cytoplasm to nucleus. The mechanism of β-catenin regulation of EMT and VM remains to be further studied.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eEGFR-regulated EMT is a driver of vasculogenic mimicry in Nasopharyngeal Carcinoma.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e: (I) Conception and design: X Peng, X Liu; (II) Administrative support: X Liu, B Yu; (III) Provision of study materials or patients: Y Lou; (IV) Collection and assembly of data: Y Yue, H Feng; (V) Data analysis and interpretation: Y Yue; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll clinical studies were approved by the Ethics Committee of Southern Medical University (Ethnical approval No. NFEC-2017-165). The study protocol is performed in accordance with the relevant guidelines.\u0026nbsp;This study informed consent was obtained from all subjects and/or their legal guardian(s).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by grants from the National Natural Science Foundation of China (81702696), Natural Science Foundation of Guangdong Province of China (2017A030310040 and 2020A1515010176), and Supported by Beijing xisike Clinical Oncology Research Foundation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.Y. and P.X.H. designed the study. Y.Y., L.Y.F., F.H.R. and Y.B.L. performed the work. Y.Y., L.X. and P.X.H. performed the statistical analysis. D.R., L.B.J., H.H.R. and Y.X.F reviewed the data. All authors discussed the data. Y.Y, P.X.H, Y.B.L and L.X.drafted the manuscript. All co-authors read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThanks to BIOTECH PHARMACEUTICAL CO.LTD for providing Nimotuzumab injection for the experiment.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHern\u0026aacute;ndez De La Cruz, O.N., et al., Regulation Networks Driving Vasculogenic Mimicry in Solid Tumors. Frontiers in Oncology, 2020. 9.\u003c/li\u003e\n\u003cli\u003eKrishna Priya, S., et al., Tumour angiogenesis-Origin of blood vessels. International Journal of Cancer, 2016. 139(4): p. 729-735.\u003c/li\u003e\n\u003cli\u003eManiotis, A.J., et al., Vascular channel formation by human melanoma cells in vivo and in vitro: vasculogenic mimicry. Am J Pathol, 1999. 155(3): p. 739-52.\u003c/li\u003e\n\u003cli\u003eSeftor, R.E.B., et al., Tumor Cell Vasculogenic Mimicry. The American Journal of Pathology, 2012. 181(4): p. 1115-1125.\u003c/li\u003e\n\u003cli\u003eAndreucci, E., et al., Physicochemical aspects of the tumour microenvironment as drivers of vasculogenic mimicry. Cancer and Metastasis Reviews, 2022.\u003c/li\u003e\n\u003cli\u003eLezcano, C., et al., Merkel cell carcinoma expresses vasculogenic mimicry: demonstration in patients and experimental manipulation in xenografts. Lab Invest, 2014. 94(10): p. 1092-102.\u003c/li\u003e\n\u003cli\u003eBhuniya, A., et al., Tumor activated platelets induce vascular mimicry in mesenchymal stem cells and aid metastasis. Cytokine, 2022. 158: p. 155998.\u003c/li\u003e\n\u003cli\u003eSong, X., et al., Microbial metabolite deoxycholic acid promotes vasculogenic mimicry formation in intestinal carcinogenesis. Cancer Science, 2022. 113(2): p. 459-477.\u003c/li\u003e\n\u003cli\u003eCarroll, C.P., et al., Targeting hypoxia regulated sodium driven bicarbonate transporters reduces triple negative breast cancer metastasis. Neoplasia, 2022. 25: p. 41-52.\u003c/li\u003e\n\u003cli\u003eRiemann, A., et al., Extracellular Acidosis Modulates the Expression of Epithelial-Mesenchymal Transition (EMT) Markers and Adhesion of Epithelial and Tumor Cells. Neoplasia, 2019. 21(5): p. 450-458.\u003c/li\u003e\n\u003cli\u003eRiemann, A., et al., The Acidic Tumor Microenvironment Affects Epithelial-Mesenchymal Transition Markers as Well as Adhesion of NCI-H358 Lung Cancer Cells. Adv Exp Med Biol, 2021. 1269: p. 179-183.\u003c/li\u003e\n\u003cli\u003eLamouille, S., J. Xu and R. Derynck, Molecular mechanisms of epithelial\u0026ndash;mesenchymal transition. Nature Reviews Molecular Cell Biology, 2014. 15(3): p. 178-196.\u003c/li\u003e\n\u003cli\u003eZhao, J., et al., LncRNA PVT1 induces aggressive vasculogenic mimicry formation through activating the STAT3/Slug axis and epithelial-to-mesenchymal transition in gastric cancer. Cellular Oncology, 2020. 43(5): p. 863-876.\u003c/li\u003e\n\u003cli\u003eLi, W. and Y. Zhou, LRIG1 acts as a critical regulator of melanoma cell invasion, migration, and vasculogenic mimicry upon hypoxia by regulating EGFR/ERK-triggered epithelial\u0026ndash;mesenchymal transition. Bioscience Reports, 2019. 39(1).\u003c/li\u003e\n\u003cli\u003eChen, Y.P., et al., Nasopharyngeal carcinoma. Lancet, 2019. 394(10192): p. 64-80.\u003c/li\u003e\n\u003cli\u003eLuo, Y., et al., Foxq1 promotes metastasis of nasopharyngeal carcinoma by inducing vasculogenic mimicry via the EGFR signaling pathway. Cell Death \u0026amp; Disease, 2021. 12(5).\u003c/li\u003e\n\u003cli\u003eLuo, Q., et al., Vasculogenic mimicry in carcinogenesis and clinical applications. Journal of Hematology \u0026amp; Oncology, 2020. 13(1).\u003c/li\u003e\n\u003cli\u003eHuang, Y., W. Hong and X. Wei, The molecular mechanisms and therapeutic strategies of EMT in tumor progression and metastasis. Journal of Hematology \u0026amp; Oncology, 2022. 15(1).\u003c/li\u003e\n\u003cli\u003eLiu, Q., et al., The relationship between vasculogenic mimicry and epithelial-mesenchymal transitions. Journal of Cellular and Molecular Medicine, 2016. 20(9): p. 1761-1769.\u003c/li\u003e\n\u003cli\u003eNusse, R. and H. Clevers, Wnt/beta-Catenin Signaling, Disease, and Emerging Therapeutic Modalities. Cell, 2017. 169(6): p. 985-999.\u003c/li\u003e\n\u003cli\u003eLuo Y, Wang J, Wang F, Liu X, Lu J, Yu X, Ma X, Peng X, Li X: \u003cstrong\u003eFoxq1 promotes metastasis of nasopharyngeal carcinoma by inducing vasculogenic mimicry via the EGFR signaling pathway\u003c/strong\u003e. \u003cem\u003eCell death \u0026amp; disease \u003c/em\u003e2021, \u003cstrong\u003e12\u003c/strong\u003e(5):411\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Nasopharyngeal carcinoma, EGFR, Vasculogenic mimicry EMT","lastPublishedDoi":"10.21203/rs.3.rs-2220879/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2220879/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eVascular mimicry is emerging as a potential target for anti-tumorigenesis.It involves the formation of microvascular channels composed of tumor cells. However, the mechanism of how tumor cells build into microvascular channels is not clear.The existence of a relationship between EMT and VM has been reported in the literature but the exact regulatory mechanism is unclear. Whether EMT regulates VM formation and its specific mechanism need to be further verified in NPC\u003c/p\u003e\u003ch2\u003eMaterials and Methods\u003c/h2\u003e \u003cp\u003eWe detected the relationship between EMT indicators and VM by immunohistochemical experiments. Also, the relationship between EMT indexes and VM indexes and clinical staging was analyzed. Cellular assays and immunoprotein blotting assays were used to detect EMT and VM changes in cells after addition of EGFR inhibitors. VM and EMT indices were examined after EGFR-targeted drug treatment in a subcutaneous tumorigenesis assay in nude mice.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eEGFR-regulated EMT is a driver of vasculogenic mimicry in Nasopharyngeal Carcinoma.\u003c/p\u003e","manuscriptTitle":"EGFR-regulated EMT is a driver of vasculogenic mimicry in Nasopharyngeal Carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-11-21 15:31:36","doi":"10.21203/rs.3.rs-2220879/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c3417066-086d-401f-b19b-21320c9382b3","owner":[],"postedDate":"November 21st, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-01-11T04:59:07+00:00","versionOfRecord":[],"versionCreatedAt":"2022-11-21 15:31:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2220879","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2220879","identity":"rs-2220879","version":["v1"]},"buildId":"cBFmMYwuxLRRLfASyISRj","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}